1. Field of Invention
The present invention relates to the field of spectroscopy and spectrophotometers. Specifically the invention relates to the field of ultraviolet, visible, and infrared spectroscopy. More specifically the invention relates to the field of circular polarized light spectroscopy. The invention is a new spectrometer that uses circular polarized light to generate a circular dichroism spectrum free from interference.
2. Description of Related Art
Spectroscopy is the science and application of light measurement. A spectrometer or spectrophotometer is the instrument that is used to measure the spectrum of a substance. A spectrometer has a light source, a light selection device, a sample compartment, and a light detector along with appropriate electronic and computer controls and data acquisition capabilities. Common scientific spectrometers have a light source that can generate light in the ultraviolet (UV), visible, and infrared (IR) regions. A common UV light source is the hydrogen or deuterium lamp. A visible light source is usually a tungsten lamp. An IR source is commonly a special ceramic material that is heated to a given temperature.
A light selection device is usually an arrangement of slits, filters, and diffraction gratings and other elements that allow the selection of light with particular characteristics to proceed through the optical configuration. The characteristics selected for could be the wavelength or the polarization or both. The wavelength selection device can select a very narrow range of wavelengths from the incoming polychromatic light. If the wavelength selection device is good enough, the light coming from the device is virtually monochromatic light. This type of wavelength selection characterizes the dispersive spectrometer.
An alternate configuration for a wavelength selection device, termed Fourier-transform spectrometer, makes use of the Michelson interferometer and computer manipulations of the resulting signal to generate an absorption spectrum. A beam splitter splits the beam from the light source. One of the two resulting beams of light is reflected from a fixed mirror back to the beam splitter and the second beam of light is reflected from a movable mirror back to the beam splitter. The beam splitter recombines the light from the two reflective mirrors to form a single beam that goes through the rest of the components of the spectrometer. Because the two light paths are identical only at one instance in time, an interference pattern versus time is generated. Computer manipulations of the resulting signal from the interference pattern result in an absorption spectrum This Fourier-transform spectrophotometer has become the instrument of choice in many situations because higher light levels are transmitted through the instrument which gives a better signal to noise ratio in the resulting spectrum.
The particular light selected is used to probe the sample, which can be liquid, solid, or gas, and is detected at a light detector that is usually a photomultiplier or photodiode with appropriate electronic amplification and recording devices. Another light modifying element of a light selection device is a polarization modulator (PM). A PM has the ability to take linear polarized light and modulate it at a fixed modulation frequency between right circular polarized (RCP) and left circular polarized (LCP) light. A PM has an optical element, such as fused silica, and an attached transducer for vibrating the optical element at a particular frequency as described in U.S. Pat. No. 5,652,673. As the optical element vibrates under the influence of the transducer, the optical element is compressed and extended in an oscillating fashion. The effect of this oscillation in the optical element is to cause the light that leaves the element to be modulated between LCP and RCP. When a beam of incident radiation modulated between RCP and LCP is used to probe a sample, the sample may absorb selectively the RCP or the LCP light. If the wavelength of the incident light is controlled so that the entire spectrum of interest can be sampled as a probe, the light hitting the light detector will be a function of the difference in the ability of the sample to absorb LCP and RCP light at the various wavelengths that are selected. When the signal from the light detector is demodulated at the same frequency that the PM is operating, a spectral scan can be obtained that shows the difference between the LCP and the RCP light absorbed by the sample as a function of the wavelength of the incident circular polarized beam of light This differential spectral scan is called the circular dichroism (CD) spectrum of the sample. The CD spectrum of a material can be used to probe the chiral properties of a material, and, thus, it is very important in the understanding of the absolute molecular configuration of chemical compounds.
A carbon atom can have four different atoms or groups of atoms covalently attached to it. The attached groups form a tetrahedron that, if the groups are not identical, can have either an R or an S configuration. This asymmetrical configuration in the molecular structure of the compound gives rise to the differential absorption of the LCP and the RCP light. If equal concentrations of the R configuration and the S configuration are present, the sample is termed a racemic mixture of the two configurations. Because the equal concentrations of R and S configurations will absorb the RCP and LCP light equally, there will be no CD spectrum of the sample. If only a single configuration of a molecule is present in the sample, the sample will give a CD spectrum. If, for example, the R configuration is present as 75% of the sample and the S configuration is present as 25% of the sample, the CD spectrum will have the pattern of the R configuration but will not have the full intensity of a sample of the pure R configuration of the molecule. In this manner the chiral purity of a sample can be determined. In certain drugs, only one of the two possible configurations gives the desired effect. If the CD spectrum of a chemical compound can be accurately determined it can be compared to theoretical calculations to test the accuracy of the theoretical understanding of the chemical compound.
The optical configuration of a spectrometer described above, and shown in FIG. 1, can be represented by the following symbol pattern:
LSxe2x86x92Gxe2x86x92Pxe2x86x92PMxe2x86x92Sxe2x86x92Dxe2x80x83xe2x80x83(I)
where LS is the light source 2; G is a wavelength selection device or Michelson interferometer 4; P is a linear polarizer 6, needed to define a single state of polarization such as vertical polarized light; PM is a polarization modulator 8 with stress axis at 45xc2x0 from the axis of the linear polarizer, which switches the polarization between LCP and RCP states; S is the sample 10 and D is the detector 12. An example of a G is the Fourier transform infrared interferometer sold by Bomem of Quebec, Canada. An example of a P is an aluminum wire-grid infrared polarizer from Specac Inc., Smyrna, Ga. An example of a D is a mercury cadmium telluride detector from EGandG Optoelectronics in Santa Clara, Calif. An example of a PM is the photoelastic modulator sold by Hinds Instruments in Hillsboro, Oreg. In practice the PM switches between LCP and RCP at a rate of between 20 and 100 kilohertz.
The intensity of the light that strikes the detector can be represented by equation number 1.
ID=TR+CDxe2x80x83xe2x80x83(1)
where ID 14 is the intensity at the detector and TR 20 is the ordinary transmitted radiation spectrum of the sample with an absorbance, A. TR is the amount of light that passes through the sample and reaches the light detector. A sample will absorb some of the light at any particular wavelength of light, and that is termed the absorbance of the sample. The absorbance, A, is defined as the negative logarithm of the base 10 of the ratio of the intensity at the detector when the sample is in place, TR, divided by the same intensity when the sample has been removed, TR0. This is given by
A=xe2x88x92log10(TR/TR0)xe2x80x83xe2x80x83(2)
The absorbance of a sample will vary as a function of the wavelength and concentration of the absorbing compound in the sample compartment. The light that is not absorbed by the sample is the light that is transmitted through the sample and is termed the transmission spectrum of the sample.
The CD term 18 of the equation (1) is that part of the detector signal, ID 14, that oscillates at the PM modulation frequency. At any given wavelength, the CD term could add to, subtract from, or not affect the TR term 20 of equation (1). The CD term, which is obtainable only at the PM modulation frequency, can be considered a change in absorbance of the sample at the PM modulation frequency. The CD term is the difference between amount of LCP light absorbed by the sample and the amount of RCP light absorbed by the sample. Thus if more LCP light is absorbed, the CD term will be positive and if more RCP light is absorbed, the CD term will be negative. The TR term is very large compared to the CD term in equation (1). Typically for determinations of the CD spectra in the infrared region of the spectrum, the TR term is ten thousand to one hundred thousand as strong as the CD signal. However, the CD term can be observed in practice because the signal from the detector that oscillates in frequency with the PM frequency can be isolated from the rest of the signal by a lock-in amplifier, LIA 16. An example of a LIA is the Model SR810 lock-in amplifier from Stanford Research Systems, Sunnyvale, Calif.
The measured circular dichroism spectrum, xcex94A 24 is defined as the absorbance for LCP light, AL, minus the absorbance for RCP light, AR, as
xcex94A=ALxe2x88x92ARxe2x80x83xe2x80x83(3)
To obtain the circular dichroism spectrum, xcex94A 24 the CD term 18 is divided by the TR term 20 by means of a software operation DIV 22, and then multiplied by a software calibration factor, CAL, as
xcex94A=CAL(CD/TR)xe2x80x83xe2x80x83(4)
In an optical configuration as shown in I, an unwanted background spectrum occurs and is given the technical term linear birefringence (LB). This is an unwanted background (UB) that disturbs the zero base line upon which the desired CD spectrum appears. LB can be represented as a part of an optical configuration and as shown in FIG. 2 as an optical-electronic diagram:
LSxe2x86x92Gxe2x86x92P1xe2x86x92PMxe2x86x92LBxe2x86x92P2xe2x86x92Dxe2x80x83xe2x80x83(IIa)
or
xe2x80x83LSxe2x86x92Gxe2x86x92P1xe2x86x92LBxe2x86x92PMxe2x86x92P2xe2x86x92Dxe2x80x83xe2x80x83(IIb)
where LB is the source of the linear birefringence 26 with axes parallel or perpendicular to those of the PM. The LB may be present as a birefringent plate, strain in the sample windows, or strain in the PM. In practice, the precise cause of LB is very difficult to define. A second polarizer, P2 28, parallel, perpendicular or some angle between, to the first polarizer, P1 6, has been added to the optical configuration prior to the detector. The second polarizer may be a linear polarizer inserted into the configuration intentionally or the linear polarization intensity of the detector itself. Thus the LB cannot be eliminated from the system. The mathematical expression for the intensity of the radiation at the detector, ID 30, is given by equation (5):
ID=TRxe2x80x2+UBxe2x80x83xe2x80x83(5)
where the TRxe2x80x2 term 34 is closely related to the TR term 20 in equation (1), and the UB term 32 is the signal that represents an unwanted background due to the linear bireflingence in the optical path between the two polarizers, P1 6 and P2 28. The final circular dichroism spectrum due to the unwanted background in the optical path is given by xcex94AB 36,
xcex94AB=CAL(UB/TRxe2x80x2)xe2x80x83xe2x80x83(6)
where the UB signal is divided by the TRxe2x80x2 and then calibrated as in equation (4).
An alternative optical configuration for a CD spectrophotometer interchanges the position of the sample S 10 and the polarization modulator PM 8 from configuration I FIG. 2. The addition of P2 28 to the configuration gives configuration III, as shown below and as illustrated in the optical-electronic diagram in FIG. 3,
LSxe2x86x92Gxe2x86x92P1Sxe2x86x92PMxe2x86x92P2xe2x86x92Dxe2x80x83xe2x80x83(III)
The mathematical expression for the intensity of the light hitting the detector ID 38 for configuration m is given by the equation
ID=TRxe2x80x2xe2x88x92CD/2.xe2x80x83xe2x80x83(7)
Here, the TRxe2x80x2 term 42 is similar to the term in equation (5) because the presence of P2 28 in the optical configuration, and the CD term 40 has the opposite sign and one-half of its value in equation (1). The opposite sign of the CD term arises because the PM is positioned after the sample instead of before the sample. The final circular dichroism spectrum 44 is obtained by division ofxe2x88x92CD/2 by TRxe2x80x2 followed by calibration as
xe2x88x92xcex94A/2=CAL((xe2x88x92CD/2)/TRxe2x80x2)xe2x80x83xe2x80x83(8)
All of the above information is well known in the prior art, and CD spectrophotometers have been manufactured using the above configurations. Spectrometers made by Jasco, Aviv, and Olis are available that obtain CD spectrum in the ultraviolet and visible region. Bomem/BioTools manufactures instruments that can obtain a CD spectrum in the infrared region.
Current CD spectroscopy is faced with separating the UB contribution to the spectrum from the desired CD part of the spectrum. This is currently done by collecting the spectrum with and without the sample in place, storing the spectra in a digital format, and mathematically subtracting two spectra. Although informative, the operation cannot be ideal because introduction of the sample into the optical beam creates new UB functions. The current invention eliminates UB without any subsequent optical measurement or stored blank spectrum, and thus obtains for the first time a CD signal in real time that is free of UB interference.
The introduction of a second PM into the optical configuration eliminates the UB from the CD spectrum of a sample. The pure CD spectrum produced by the current invention gives a heretofore unobtainable precise and accurate CD spectrum of a sample in a single measurement. The improved CD spectrum of a sample can be used to investigate basic scientific questions about the sample such as absolute configuration, optical purity, and structural conformation.